摘要
目的:探讨小干扰RNA(siRNA)介导的核糖核苷酸小亚基M2(ribonucleotide reductase M2,RRM2)沉默对顺铂(DDP)诱导的卵巢癌耐药细胞株SKOV3/DDP敏感性的影响。方法:采用间歇浓度梯度递增法诱导SKOV3/DDP;将RRM2基因的特异性siRNA转染SKOV3/DDP细胞,另设空白组和SKOV3/DDP-RRM2-neg(阴性组)做为对照;荧光PCR技术和蛋白质印迹法分别检测转染后各组细胞中RRM2基因mRNA和蛋白表达;MTT法检测RNAi对SKOV3/DDP细胞药物敏感性的影响。结果:成功诱导出卵巢癌DDP耐药细胞株SKOV3/DDP细胞,其耐药系数达到3.6,属低度耐药,但对吉西他滨(Gem)仍敏感。DDP对干扰组、阴性组和空白组细胞48h的IC50分别为(3.09±0.11)、(9.88±0.37)和(10.35±0.03)μg/mL,3者间差异有统计学意义,P<0.001。干扰组细胞对DDP的敏感度提高了约3倍,其RF为3.3。SKOV3/DDP-RRM2-RNAi 667(Ⅰ)组RRM2 mRNA水平下调为74.1%,P=0.010;SKOV3/DDP-RRM2-RNAi480(Ⅱ)组RRM2 mRNA水平下调为55.9%,P=0.016;SKOV3/DDP-RRM2-RNAi1179(Ⅲ)组RRM2mRNA水平下调为43.1%,P=0.001。其中,以转染Ⅰ组基因沉默率(82.33%)最高,P<0.001;阴性组(0.008 6%)与空白组(0.008 2%)比较,差异无统计学意义,P=0.133。转染RRM2的不同靶序列72h后RRM2蛋白的表达量最低,Ⅰ组为0.062±0.006,Ⅱ组为0.314±0.002,Ⅲ组为0.123±0.002,与阴性组(0.715±0.034)和空白组(0.516±0.040)比较均明显降低,但以转染Ⅰ组细胞的蛋白相对表达量下降最明显,F=658.6,P=0.003 3。Gem和DDP联合作用72h时,转染组细胞凋亡率为(96±3.0)%,与其各组比较,差异均有统计学意义,P值均<0.001。结论:通过RNAi技术可有效抑制RRM2基因在卵巢癌中的转录和翻译水平,增加DDP耐药细胞的药物敏感性,并促进DDP诱导的卵巢癌耐药细胞凋亡,在一定程度上逆转DDP耐药性;RNAi技术联合Gem及DDP作用可有效提高卵巢癌DDP耐药细胞的凋亡率,有望成为铂类耐药的卵巢癌晚期患者一线治疗方案。
OBJECTIVE：To investigate the effect of small interference RNA （siRNA） -mediated the M2 subunit of ribonucleotide reductase （RRM2） silencing on the drug sensitivity of cisplatin-resistant SKOV3/cisplatin （DDP） （ovarian carcinoma） cells. METHODS： Cisplatin-resistant ovarian carcinoma cell strain SKOV3/DDP was induced intermittently with increasing drug dosage, siRNA transfection was mediated by lipofectamine 2000 to silence RRM2. Un-transfected cell served as negative controls, mRNA and protein expression levels of RRM2 were evaluated by real-time PCR and western blot after transfection. The cell growth inhibition rate was evaluated by MTT. RESULTS： Stable cisplatin-resistant SK- OV3/DDP cell strain was established with the resistance indexes of SKOV3/DDP for 3.6, which still sensitive to theDem. At 48 h,the ICs0 of SKOV3/DDP-RRM2-RNAi was （3.09±0.11） μg/mL which was obviously lower than SKOV3/ DDP-RRM2-neg （9.88±0. 37） μg/mL and SKOV3/DDP （ 10. 35 ± 0. 03） μg/mL （P 〈 0. 001 ）. The RF of SKOV3/ DDP-RRM2-RNAi was 3.3. RRM2 mRNA of SKOV3/DDP-RRM2-RNAi667（ I ） decreased to 74.1% （P=0. 010） ,SK- OV3/DDP-RRM2-RNAi480（ Ⅱ） decreased to 55.9% （P=0. 016） and SKOV3/DDP-RRM2-RNAil179（ Ⅲ） decreased to 43.1% （P = 0. 001）. The RRM2 silencing rate of SKOV3/DDP-RRM2-RNAi667 （ Ⅰ ） was the highest （82.33 %, P〈 0. 001）. Silencing rate in SKOV3/DDP-RRM2-neg （0. 008 6%） and SKOV3/DDP （0. 008 2%） didn＇t have significant difference（P=0. 133）. The protein level was the lowest at 72 h,SKOV3/DDP-RRM2-RNAi667（ I ） was 0. 062±0. 006, SKOV3/DDP-RRM2-RNAi480（ Ⅱ） was 0. 314 ± 0. 002 and SKOV3/DDP-RRM2-RNAi1179 （ Ⅲ ） was 0. 123±0. 002. Compared with SKOV3/DDP-RRM2-1ipo and SKOV3/DDP-RRM2-neg cell,the RRM2 protein expression level decreased in all three cells and heaviest in SKOV3/DDP-RRM2-RNAi667（ I ） （F=658.6 ,P=0. 003 3）. After 24 h of transfection, the highest rate of apoptosis was （96 ±3.0）%, which appeared at 72 h. The difference of apoptosis rate in SKOV3/ DDP-RRM2-RNAi＋DDP-t-Gem at different time points were statistically significant （P= 0. 003）,when compared with SKOV3/DDP-RRM2-RNAi, SKOV3/DDP-RRM2-RNAi＋ DDP and SKOV3/DDP-RRM2-RNAi -I- Gem. CONCLUSIONS ： RNA interference silenced RRM2 gene in SKOV3/DDP cell line significantly,which reversed cell resistance to DDP and promoted ovarian cancer cells apoptosis rate induced by cisplatin. RRM2 probably plays an important role in cell resistance to DDP in ovarian cancer. RNAi technology combinded with gemcitabine and cisplatin can effectively improve the apoptosis rate of the cisplatin-resistant ovarian cancer cell,which is expected to become the first-line treatment option for the eispla- tin-resistant ovarian cancer.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2014年第4期273-279,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
青岛市科技计划基础研究项目〔No.11-2-4-2-(10)-jch〕
关键词
卵巢肿瘤
基因沉默
RNA干扰
耐药逆转
核糖核苷酸小亚基M2
ovarian neoplasms
gene silencing
RNA interference
ribonucteotide reductase M2
reversal of cisplatinresistance
